Dot blot assays were set up with the antiserum, and consistent with Western blot results, the antiserum cannot recognize BEFV particles (Fig. When the antiserum produced was first evaluated by Western blotting, while it reacted with rG1, the antiserum, unexpectedly, produced no signals against BEFV particles (Fig. The estimated molecular weight of the recombinant G1 protein (rG1) was 42 kDa, and the antigenicity of rG1 was confirmed by Western-blotting using the previously produced antiserum raised against purified BEFV particles (Fig. coli expression system, the BEFV G1 region was expressed for antiserum production (Fig. To overcome the problem of high background levels, we sought to produce antiserum with BEFV antigens from non-BHK-21 cell culture systems. coli-expressed BEFV G1 region cannot detect BEFV particles in dot blot assays Antibodies raised using these antigens were evaluated in dot blot assays.įull size image Antibodies raised against E. To determine the type of antigens that is most suitable for raising antibodies to be used in dot blot assays, three different kinds of BEFV antigens were prepared: 1) purified BEFV particles, 2) E. Therefore, the present study aimed to develop a dot blot assay for quick quantitation of BEFV particles in a cell culture preparation. Conventional methods such as TCID 50 and plaque assays are time-consuming and require days of waiting time. However, antibody titers elicited by inactivated BEFV vaccines are not long-lasting and booster vaccination is required every year, creating a stable market demand for the vaccine.ĭuring the manufacturing process of inactivated BEFV vaccine, a quick and reliable virus titering method is essential. Inactivated BEFV vaccines serve as the major tool for BEF control. G contains four antigenic regions (G1-G4), and G1 is a major region of neutralizing epitopes. N is the nucleoprotein and G is the envelope glycoprotein, a major antigen that elicits neutralizing antibodies. As a member of the genus Ephemerovirus in the family Rhabdoviridae, BEFV has a single-stranded, negative sense RNA genome that encodes five structural proteins (N, P, M, G, and L) and five nonstructural proteins. While mortality due to BEFV infection is low, reduced milk production during pandemic periods can result in significant economic losses for dairy farms. A dot blot assay kit using this antiserum can be developed as a quick and economical way for BEFV titering.īovine ephemeral fever virus (BEFV) infects cattle and water buffalos through arthropod vectors, causing fever, muscle stiffness, and ocular/nasal discharge. ConclusionsĮ.coli-expressed N protein is a suitable antigen for the production of antiserum that can detect BEFV particles with a high signal-to-noise ratio. Finally, antibodies raised against E.coli-expressed BEFV N protein detected BEFV particles with a high signal-to-noise ratio in dot blot assays. Antibodies raised against E.coli-expressed BEFV G1 region could not detect BEFV particles in dot blot assays.
Results showed that antibodies raised against purified BEFV particles can detect BEFV particles, but it also showed a high background level from the proteins of BHK-21 cells. coli)-expressed BEFV G1 region, and 3) E. Three different kinds of BEFV antigens were prepared to raise primary antibodies for BEFV detection in dot blot assays: 1) purified BEFV particles, 2) Escherichia coli ( E. We sought to develop a quick dot blot assay for BEFV titering. During the manufacturing process of inactivated vaccine for virus control, it is important to determine the virus titer, but traditional methods such as plaque assay and TCID 50 assay require days of waiting time. Bovine ephemeral fever virus (BEFV) causes fever and muscle stiffness in cattle, resulting in negative economic impact for cattle and dairy farms.
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